Immunohistochemical localization of dopamine-beta-hydroxylase in human and monkey eyes

PURPOSE: To investigate the pattern of dopamine-beta-hydroxylase (DBH)-containing fibers in human and monkey eyes. METHODS: DBH-containing fibers were detected by immunohistochemistry. The primary antibody used recognized DBH, the key enzyme in the conversion of dopamine to noradrenaline. RESULTS: In the anterior segment, DBH immunoreaction product was found in the peripheral corneal endothelium layer, in both the dilator and sphincter muscles of the iris, as well as in the anterior border layer of the iris. The ciliary muscle and the stroma of the ciliary processes were also zones of concentration. In the posterior segment, staining was seen around blood vessels in the choroid, in the vascular walls of the short posterior ciliary arteries and in the ciliary nerves. The retina was also immunopositive, with specific labeling in cones and rods of photoreceptors, inner and outer plexiform layers and ganglion cell layer. There was no significant difference in the distribution of DBH-related immunoreactivity in human and monkey eyes. CONCLUSIONS: The localization of DBH-related immunoreactivity is generally consistent with the known physiological roles of noradrenaline. However, an apparently high concentration of the enzyme in the anterior border layer of the iris and in retinal photoreceptors raises questions about the possible role of DBH-containing fibers in these structures.     

成人结膜干细胞定位特征的免疫组织化学研究

目的:研究成人结膜上皮细胞中,粘蛋白5AC(MUC5AC),角蛋白7,p63和增殖细胞核抗原(PCNA)的表达及分布情况,探讨成人结膜上皮干细胞的定位特征。方法:获取角膜移植供者的睑球结膜,采用HE免疫组化染色法,观察MUC5AC,角蛋白7,p63和PCNA的表达及分布情况。结果:PCNA及p63表达情况一致,主要表达在睑缘皮肤黏膜交界处及近角膜缘结膜上皮深层细胞。角蛋白7在穹窿部球结膜的固有层中的腺体细胞有阳性表达,MUC5AC在各部位结膜上皮的杯状细胞的胞质阳性表达。结论:MUC5AC细胞角蛋白7和p63及PCNA在成人结膜上皮细胞的特征性表达情况可以推测具有分化潜力的结膜干细胞存在于睑缘皮肤黏膜交界处及近角膜缘球结膜上皮深层细胞。     

Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes

PURPOSE: To examine immunohistochemically the localization of myocilin/trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) protein in the glaucomatous and normal trabecular meshworks. METHODS: Trabecular tissues were used from one eye with late-onset goniodysgenetic glaucoma, three with primary open angle glaucoma (one of which had the MYOC/TIGR gene mutation), two with exfoliation glaucoma and one without glaucoma. For light microscopic immunohistochemistry, frozen sections were stained by the avidin-biotin complex method using anti-MYOC/TIGR polyclonal antibody. For electron microscopic immunohistochemistry, the pre-embedding method using the same antibody was performed. Double immunostaining using both anti-MYOC/TIGR and anti-type VI collagen antibodies was done by the immunofluorescence method. RESULTS: With light microscopy, immunoreactivity was seen in the whole trabecular meshwork of each of the specimens. No notable differences were detected in staining among the types of glaucoma, or between the eyes with and those without the gene mutation. Under electron microscopy, immunoreaction products were observed not only in the cytoplasm of the trabecular cells but also in the extracellular matrix, where staining was associated with the long-spacing collagen, fine granular materials and possibly microfibrils. With double immunohistochemistry, MYOC/TIGR was colocalized with type VI collagen in the trabecular meshwork. CONCLUSIONS: In glaucomatous and normal trabecular meshworks, the MYOC/TIGR protein is distributed in the extracellular matrix colocalizing with type VI collagen.     

Microbial contamination of donor eyes

A total number of 1,516 donor eyes received from various sources during the years 1973-1985 were subjected to the isolation of bacterial contamination. The bacterial cultures taken from the pretreatment eyeball showed culture growth in 366 (24.1%) eyes. Of the 366 positive cultures, 331 (21.8%) were bacterial and 35 (2.3%) were fungal. Amongst the bacterial the major contamination was by staphylococcus aureus and albus and pseudomonas aeruginosa. Gentamicin was found to be the most sensitive antibiotic against a wider group of organisms, the next being chloramphenicol. Thus, treatment of a cadaver eye with a solution of normal saline containing 0.1-0.5 mg/ml of gentamicin is recommended before and after the donor eye is enucleated.     

Immunohistochemical demonstration of metallothionein in eyes with choroidal melanomas

PURPOSE: The since immunohistochemically detectable metallothionein (MT) overexpression has been described in a variety of human tumours, including skin melanomas, in relation to different stages of tumour development and progression. MATERIALS AND METHODS: We used a monoclonal antibody to investigate the distribution of MT in 18 formalin-fixed, paraffin-embedded, surgically enucleated eyes with choroidal melanomas, from 18 patients (8 male, 10 female; age range 30-83 years, mean 58.7). Clinico-pathological details and follow-up data (2-124 months, mean 36.1) were also available. MT immunoreactivity was recorded and the percentage of stained cells was graded for semiquantitative purposes. Correlations between immunohistochemical data and morphological characteristics of melanomas were investigated using non-parametric methods; survival analysis was done by the Kaplan-Meier method and the survival curves were compared by the Mantel-Cox log-rank test. RESULTS: MT immunoexpression was found in 15/18 cases (83.3%) with staining scores from 1 to 3; MT staining varied in intensity and was mainly localized in the cytoplasm, although a combined nuclear/cytoplasmic reactive pattern was seen in neoplastic elements. No differences in MT immunostaining were seen in relation to age or sex, tumour size, histotype and amount of pigment; univariate analysis of survival data showed no prognostic significance regarding MT expression. CONCLUSIONS: The immunohistochemical evidence of MT in neoplastic elements could be related to the production of this scavenging protein in the tumour for cell defense mechanisms against hydroxyl free radicals, and to act as a Zn donor, since Zn is required for the synthesis of DNA and DNA-repair enzymes.     

Effect of bag-in-the-lens implantation on posterior capsule opacification in human donor eyes and rabbit eyes

PURPOSE: To evaluate bag-in-the-lens implantation by studying the feasibility of implanting a new type of intraocular lens (IOL) and the occurrence of posterior capsule opacification (PCO) in human postmortem eyes and in eyes of living rabbits. SETTING: Department of Ophthalmology, University of Antwerp, Belgium, and Netherlands Research Institute of Amsterdam, Amsterdam, The Netherlands. METHODS: The IOL was implanted in 10 postmortem human donor eyes (in vitro study) and in 17 eyes of 10 rabbits (in vivo study). The postmortem capsular bags were cultured for 4 to 6 weeks, and the rabbits were killed 1 to 5 months after implantation. All capsular bags with the bag-in-the-lens were examined by light microscopy and scanning electron microscopy. RESULTS: The IOL design was highly effective in restricting lens epithelial cell (LEC) proliferation in the remaining lens bag in human donor eyes and in rabbit eyes. In eyes in which the capsules were not positioned well within the groove of the IOL, LEC proliferation and PCO occurred. CONCLUSION: Bag-in-the-lens implantation was highly effective in preventing PCO in vitro and in vivo provided the anterior and posterior capsules were secured properly in the peripheral groove of the IOL.     

Immunohistochemical localization of catalase in ocular tissue

Localization of catalase in rat and bovine ocular tissues was investigated by an immunohistochemical method. Antisera to purified catalase was raised in a rabbit. Purity of catalase was determined by gel electrophoresis and specificity of the antibody was tested by immunoblot. The immunohistochemical method revealed the presence of catalase predominantly in epithelial and endothelial structures of the eye and in the retina, except in the outer segments. Catalase was found in abundance in those structures that are frequently exposed to oxygen metabolites under physiologic conditions and in such pathologic states as intraocular inflammations. These findings thus suggest that catalase, along with other antioxidant enzymes, may offer protection against the damaging effects of hydrogen peroxide.     

Characterization of age-related macular degeneration in Indian donor eyes

Purpose:The purpose of this study was to test the reliability of fundus stereomicroscopy in postmortem eyes to assign severity of age-related macular degeneration (AMD) using the Minnesota grading and confirmation by histology using Alabama and Sarks grading scales and to assess the incidence of AMD pathology in donor eyes from a South Indian population.Methods:Eyes (199) from 153 donors (55–95 years) after obtaining fundus images were processed for histology. Fundus images were graded according to the Minnesota grading system based on drusen size, area of depigmentation, and atrophy. At least one eye from each donor displaying the AMD phenotypes were subjected to histological examination. The fundus grading was correlated with histology and the stages of AMD assigned for early AMD by the Alabama AMD grading system and for both early and advanced AMD by the Sarks classification.Results:Stereoscopic examination of the fundus found that 10 of the 153 donors had features of early AMD and 3 advanced AMD. Following histological examination, one of the early AMD eyes was reclassified as advanced AMD. Early AMD features that were observed on histology included soft drusen (>63 μm), basal laminar deposits, photoreceptor outer segment degeneration, disorganization of retinal pigment epithelium (RPE), Bruch''s membrane thickening. Advanced AMD features observed in histology are extensive atrophy of RPE, choroidal neovascularization and disciform scar formation.Conclusion:Identification of either early or advanced AMD using stereomicroscopic assessment (SMA) showed high sensitivity and specificity. However, misclassification between AMD stages can occur when only SMA is used.     

Evaluation and transplantation of corneas from pseudophakic donor eyes

PURPOSE: To evaluate the quality of corneal donor tissue from pseudophakic eyes for transplantation. METHODS: Only capsular-supported posterior chamber pseudophakia was studied. Forty-five pairs of donor eyes were assessed and evaluated by standard Minnesota Lions Eye Bank (MLEB) protocol. Thirty-three pairs were unilaterally pseudophakic with the unoperated phakic eye used as a control eye. Twelve donors were bilaterally pseudophakic. All corneas were rated for corneal clarity, epithelial defects, stromal edema, Descemet's membrane folding, endothelial cell density (ECD), and cell damage by slit-lamp examination and specular microscopy. If the corneas were not transplanted, the endothelium was vital stained with trypan blue and counterstained with alizarin red S for quantitation, localization, and visualization of cell morphology and damage. RESULTS: Sixty-eight of the 90 corneas in this study did not meet transplantation criteria. A significant difference in ECD (>22%) and in overall corneal rating was found in seven (21%) of 33 pairs of unilateral pseudophakic donors. Fourteen corneal transplants were performed using corneas from the donors in this study. Nine corneas from pseudophakic donor eyes were transplanted, resulting in one primary graft failure reported. CONCLUSION: Corneas from pseudophakic donor eyes may need more extensive evaluation for endothelial viability than is currently required by eye bank standards.     

Immunohistochemical localization of cathepsin D in ocular tissues

Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues. In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and sphincter and dilator muscles of the iris; Müller cells; ganglion cells and pigment epithelium of the retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic degradation of rod outer segments.     

Attempts of tissue typing from human donor eyes

A method for HLA-ABC typing using pigmented retinal epithelial and uveal cells of the donor eye is described and the possible implications for corneal grafting are mentioned.     

Immunohistochemical localization of angiotensin-converting enzyme, angiotensin II and AT1 receptor in human ocular tissues

We investigated the immunohistochemical distribution of 3 components of the renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE), angiotensin II (AngII) and AT1 receptor (AT1), in the human eye. ACE and AngII were localized to nonpigmented epithelial cells of the ciliary body, to endothelial and epithelial cells of the cornea, to epithelial cells of the conjunctiva and to trabecular meshwork cells in the anterior part of the eye. In the posterior part of the eye, ACE and AngII were localized to ganglion cells, some cells in the inner nuclear layer, photoreceptor cells and to endothelial cells of the retinal and choroidal vessels. The overall intensity of AT1 immunoreactivity was weak in all ocular tissues, but the main localization was in ganglion cells. As a preliminary investigation, we were able to include 2 Alzheimer's disease (AD) cases. In AD, no differences from controls were found in the cellular distribution and staining intensity of all 3 antigens. The manifold localization sites of the observed antigens point to rather generalized functions of the RAS in human ocular tissues, such as regulatory effects on neuronal cells, vessels and vitreous humor homeostasis.